EP2970905B1 - Compositions et procédés pour obtenir des cultures de cellules souches mésenchymateuses enrichies - Google Patents

Compositions et procédés pour obtenir des cultures de cellules souches mésenchymateuses enrichies Download PDF

Info

Publication number
EP2970905B1
EP2970905B1 EP14765588.0A EP14765588A EP2970905B1 EP 2970905 B1 EP2970905 B1 EP 2970905B1 EP 14765588 A EP14765588 A EP 14765588A EP 2970905 B1 EP2970905 B1 EP 2970905B1
Authority
EP
European Patent Office
Prior art keywords
cells
cultures
cell
mesenchymal
medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP14765588.0A
Other languages
German (de)
English (en)
Other versions
EP2970905A1 (fr
EP2970905A4 (fr
Inventor
Brenton John SHORT
Christopher DURONIO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Stemcell Technologies Canada Inc
Original Assignee
Stemcell Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Stemcell Technologies Inc filed Critical Stemcell Technologies Inc
Publication of EP2970905A1 publication Critical patent/EP2970905A1/fr
Publication of EP2970905A4 publication Critical patent/EP2970905A4/fr
Application granted granted Critical
Publication of EP2970905B1 publication Critical patent/EP2970905B1/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the disclosure relates to methods of enriching for mesenchymal stem cells in mesenchymal stem cell cultures from bone marrow or compact bone.
  • the disclosure also relates to media for said enrichment.
  • Bone marrow is a soft spongy tissue that resides within the hollow cavity of long bones and represents about 4% of the total body weight. It is within the BM that blood cells are produced from pluripotent hematopoietic stem cells (HSC), a process referred to as hematopoiesis. It is also within the BM and lining the wall of compact bone (CB) that mesenchymal stem cells (MSC) reside. MSC are responsible for generating various cells of the body including fibroblasts, osteoblasts, chondrocytes, adipocytes, myocytes, and endothelial cells. In addition to HSC, hematopoietic monocytes, and MSC, there are several other types of cells that reside within the BM and CB making these tissues highly heterogeneous in nature.
  • HSC pluripotent hematopoietic stem cells
  • CB compact bone
  • MSC mesenchymal stem cells
  • Bone marrow provides specific microenvironments, or niches, necessary for hematopoietic and mesenchymal stem cells, osteoblasts, endothelial and other cells to co-exist and function. It is within these niches that decisions for a stem cell to become quiescent, proliferate, differentiate, and respond to external signals take place. Recently several reports have shown that cross-talk between different stem cell niches occur to elicit a proper response in vivo (7, 8, 9, 10). It is, therefore, not surprising that BM-derived cells as well as cells lining the inner walls of CB co-exist and affect each other in in vitro culture systems.
  • MSC mesenchymal stem cell
  • CSF-1 is a cytokine that oligomerizes to CSF1R leading to trans-phosphorylation of this receptor to promote cell survival, proliferation, and differentiation of mononuclear phagocyte lineages into macrophages (4, 6). Consistent with its role in regulation of macrophage lineages, exogenous CSF-1 leads to increased production of monocytes and macrophages in mice (11), while non-functional CSF-1 (12) or CSF1R (13) mice display deficient numbers of macrophages resulting in diminished inflammatory response.
  • CSF1R adenosine triphosphate (ATP)-dependent tyrosine kinase-mediated transduction signal that ultimately directs hematopoietic monocytes to proliferate and/or differentiate.
  • ATP adenosine triphosphate
  • GW2580 [5-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl) pyrimidine-2,4-diamine] and KI20227 ⁇ N- ⁇ 4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl ⁇ -NO-[1-(1,3-thiazole-2-yl)ethyl] urea ⁇ belong to a class of small molecules that specifically inhibit CSF1R kinase activity by competing with ATP binding to CSF1R kinase (14). It has been shown that GW2580 completely inhibits human CSF1R kinase at 60 nM while remaining inactive against 26 other kinases tested (3).
  • GW2580 completely inhibited CSF-1-dependent growth of mouse myeloid cells, while a CSF-1-independent cell line, human fibroblasts, and other endothelial cells remained highly resistant to GW2580 (3).
  • Ohno et al, 2006 (Mol. Cancer Ther., 5:2634-2643 ) relates to the use of a c-fms tyrosine kinase inhibitor, Ki20227, in suppressing osteoclast differentiation and osteolytic bone destruction in a bone metastasis model.
  • Komohara et al, 2012 (Cancer Sci., 103:2165-2172 ) relates to a study in which glioma cells were co-cultured with human macrophages and presents the observation that BrdU incorporation was significantly elevated in glioma cells, and that signal transducer and activator of transcription-3 (Stat3) activation was found in both cell types.
  • Conway et al, 2005 (Proc. Natl. Acad. Sci. USA, 102:16078-16083 ) relates to a study into the in vivo inhibition of colony-stimulating-factor-1 signaling with the orally bioavailable cFMS kinase inhibitor GW2580.
  • Crespo, 2011 J. Clin. Immunol., 31:1010-1020 ) relates to the use of tyrosine kinase inhibitors to ameliorate autoimmune encephalomyelitis in a mouse model of multiple sclerosis.
  • the present inventors have shown that specific inhibition of CSF1R kinase with small molecules and other compounds with similar properties in human and mouse BM and CB cell cultures reduces the number of mature macrophages in culture, which in turn, leads to an enriched culture of MSCs. Additionally, these molecules or compounds also have effects on other non-mesenchymal cells or derived progenitors present in the culture. Overall, the use of such compounds enriches for mesenchymal cells while removing or inhibiting the presence of differentiated non-mesenchymal cells.
  • the present invention is defined in the accompanying claims and relates to an in vitro method of culturing cells and cell culture media comprising a CSF1R kinase inhibitor. Embodiments of the invention are described in the following numbered paragraphs:
  • the present disclosure relates to a method of culturing cells of the mesenchymal cell lineage, said method comprising contacting the cells with a culture medium comprising a CSF1R kinase inhibitor.
  • the disclosure also relates to a method of culturing cells from bone marrow and/or compact bone to enrich the cell culture with cells of the mesenchymal cell lineage comprising contacting the cell culture with a culture media comprising a CSF1R kinase inhibitor.
  • the method comprises:
  • the method further comprises c) obtaining a population of cells enriched for cells of the mesenchymal cell lineage.
  • the CSF1R kinase inhibitor is GW2580, KI20227, HY-13075, cFMS Receptor Inhibitor II, cFMS Receptor Inhibitor III, cFMS Receptor Inhibitor IV or ARRY-382.
  • the harvested cells further comprise non-mesenchymal cells.
  • the non-mesenchymal cells are macrophages.
  • the method is performed in vitro.
  • the cells of the mesenchymal cell lineage comprise at least one of a mesenchymal stem cell, a mesenchymal cell progenitor and a stromal-derived cell.
  • the tissue sample comprises bone marrow, compact bone, adipose tissue, or any tissue where MSCs reside.
  • the cells are contacted with the culture media for at least one hour, at least one day or at least one week.
  • the concentration of the cells of the mesenchymal cell lineage is at least 10 cells/cm 2 .
  • the cells of the mesenchymal cell lineage retain their ability to form adipogenic, chrondrogenic and osteogenic cell lineages.
  • the cells from bone marrow and/or compact bone comprise cells of the mesenchymal cell lineage and non-mesenchymal cells.
  • the disclosure further relates to a cell culture media useful for culturing cells of the mesenchymal cell lineage and/or enriching cells of the mesenchymal cell lineage.
  • the cell culture media comprises a CSF1R kinase inhibitor.
  • the CSF1R kinase inhibitor is a small molecule that inhibits CSF1R kinase activity by competing with ATP binding to CSFR1 kinase.
  • the CSF1R kinase inhibitor is GW2580, KI20227, HY-13075, cFMS Receptor Inhibitor II, cFMS Receptor Inhibitor III, cFMS Receptor Inhibitor IV or ARRY-382.
  • the medium comprises 1-10 ⁇ M GW2580.
  • the media further comprises at least one growth factor.
  • the at least one growth factor is selected from the list consisting of FGF, EGF and IGF.
  • the media comprises a basal media.
  • the basal media is selected from Dulbecco's modified Eagles's medium (DMEM), advanced DMEM, BiogroTM, SkGMTM, Ham's F10, Ham's F12, Iscove's modified Dulbecco's medium, neurobasal medium, RPMI 1640 and MCDB120 medium.
  • the media further comprises a supplement.
  • the supplement is selected from:
  • the media further comprises a lipid.
  • the lipid is at least one of arachidonic acid, cholesterol, DL- ⁇ -tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitoleic acid, palmitic acid and stearic acid.
  • Table 1 shows that addition of GW2580 to CFU-F cultures of BM- or CB-MSCs led to an increase in the number of MSC colonies in culture.
  • Three assays were performed. In assay 1 the concentration of GW2580 used was 1uM, whereas in assay 2 and 3, GW2580 was used at a concentration of 2.5 uM. In all assays, cultures exposed to either concentration of GW2580 for 14 days showed an overall minimal increase of 20% in the number of MSC colonies when compared to control cultures.
  • Table 2 shows that exposure of cultures of BM- and CB-MSC to 2.5 uM GW2580 led to a 26% (0.75 to 1.01-fold) and a 310% (0.8 to 2.46-fold) increase, respectively, in the number of CD45 - /CD29 + /Sca1 + MSCs when compared to control cultures as demonstrated by flow cytometry.
  • MSC mesenchymal stem cell
  • the disclosure provides a method of culturing cells of the mesenchymal cell lineage, said method comprising contacting the cells with a culture medium comprising a CSF1R kinase inhibitor.
  • the disclosure also provides a method of culturing cells from bone marrow and/or compact bone to enrich the cell culture with cells of the mesenchymal cell lineage comprising contacting the cell culture with a culture media comprising a CSF1R kinase inhibitor.
  • the disclosure provides a method of culturing cells of the mesenchymal cell lineage comprising:
  • MSC meenchymal stem cell
  • MSCs can be isolated from any tissue of the body including, but not limited to, compact bone, adipose tissue, cord blood, peripheral blood, fallopian tube, fetal liver and lung.
  • MSCs are multipotent cells found in the stroma lining of organs that are able to differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, tenocytes, myotubes, neural cells, and hematopoietic-supporting stroma cells.
  • cell of the mesenchymal cell lineage refers to a cell that originated from a mesenchymal (stem or progenitor) cell.
  • Cells of the mesenchymal lineage include mesenchymal stem cells, mesenchymal cell progenitors (a non-hematopoietic stem cell capable of differentiating into a mesenchymal stem cell) and stromal-derived cells.
  • Stromal cells include all the different supporting cell types found in a given tissue or organ and are distinguished from the functional elements of the tissue or organ (the "parenchymal cells").
  • macrophage refers to a cell that functions in the phagocytosis of dying or dead cells and cellular debris. Macrophages are produced by the differentiation of monocytes in tissues. They are commonly found in bone marrow and compact bone. Mature phagocytes may be identified by a C45+/CD11b+ phenotype.
  • tissue sample may be any sample of tissue that contains a cell of the mesenchymal cell lineage.
  • the tissue sample may be obtained from any mammal, including, but not limited to, humans and mice.
  • the tissue sample is a bone marrow sample, a compact bone sample or a sample of adipose tissue.
  • Preferred tissues for obtaining cells of the mesenchymal cell lineage in mice include bone marrow and compact bone.
  • Preferred tissue for obtaining cells of the mesenchymal cell lineage in humans include bone marrow and adipose tissue.
  • harvesting cells refers to isolating or extracting cells from a tissue sample such as bone marrow or compact bone. Methods of harvesting cells from tissue samples are well known in the art.
  • Cells harvested from bone marrow, compact bone (for example, cells lining the wall of compact bone) and adipose tissue contain mesechymal stem cells. However, the harvested cells are often contaminated with additional, unwanted cell types. Accordingly, cells harvested from bone marrow, compact bone and adipose tissue may also contain macrophages and cells of non-mesenchymal origin such as such as hematopoietic cells and fibroblasts.
  • the present inventors discovered that inhibitors of CSF1R kinase promotes the expansion and proliferation of cells of the mesenchymal cell lineage in cell cultures obtained from bone marrow and compact bone samples. Accordingly, the present methods include contacting cell cultures with culture medium comprising a CSF1R kinase inhibitor.
  • CSF1R kinase inhibitor includes any agent, compound, small molecule or biologic that inhibits the activity of CSF1R.
  • Colony stimulating factor-1 is a cytokine that signals through the tyrosine kinase colony stimulating factor 1 receptor (CSF1R) to stimulate cell survival, proliferation, and differentiation of mononuclear phagocytes, or macrophages.
  • a CSF1R inhibitor is a small molecule that specifically inhibits CSF1R kinase activity by competing with ATP binding to the CSF1R kinase receptor thereby preventing macrophage survival, proliferation, and differentiation.
  • the CSF1R inhibitor is GW2580 [5-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine] or KI20227 ⁇ N- ⁇ 4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl ⁇ -N0-[1-(1,3-thiazole-2-yl)ethyl]urea ⁇ .
  • CSF1R inhibitors include HY-13075 ⁇ 4-cyano-N-[4-(4-methylpiperazin-1-yl)-2-(4-methylpiperidin-1-yl)phenyl]-1H-pyrrole-2-carboxamide ⁇ , cFMS Receptor Inhibitor II ⁇ 4-(3,4-Dimethylanilino)-7-(4-pyridyl)quinoline-3-carboxamide ⁇ , cFMS Receptor Inhibitor III ⁇ 4-(3,4-Dimethylanilino)-7-(4-(methylsulfonyl)phenyl)quinoline-3-carboxamide ⁇ , cFMS Receptor Inhibitor IV ⁇ 5-Cyano-N-(2,5-di(piperidin-1-yl)phenyl)furan-2-carboxamide, CSF-1 Receptor Inhibitor IV ⁇ and ARRY-382.
  • CAS numbers for a number of the compounds listed above are as follows: Inhibitor CAS Number GW2580 870483-87-7 KI20227 623142-96-1 HY-13075 885704-21-2 cFMS Receptor Inhibitor II 959860-85-6 cFMS Receptor Inhibitor III 959861-21-3 cFMS Receptor Inhibitor IV 959626-45-0
  • the expression "contacting the harvested cells with a culture media” refers to any means by which the harvested cells are cultured or incubated in a culture media.
  • the term "culture media” refers to media designed to support the growth of cells, in particular cells of the mesenchymal cell lineage.
  • Various culture media are known in the art.
  • the culture media is a basal media such as Dulbecco's modified Eagles's medium (DMEM), advanced DMEM, BiogroTM, SkGMTM, Ham's F10, Ham's F12, Iscove's modified Dulbecco's medium, neurobasal medium, RPMI 1640 and MCDB120 medium.
  • the medium may contain serum or be serum-free.
  • the harvested cells are contacted with a culture media comprising a CSF1R inhibitor for at least an hour, at least one, three or five days, at least a week or more than one week.
  • the harvested cells are contacted with a culture media comprising a CSF1R inhibitor for at least one, two, three, four or five passages.
  • the harvested cells are continuously contacted with a culture media comprising a CSF1R inhibitor for at least an hour, at least one, three or five days, at least a week or more than one week or for at least one, two, three, four or five passages.
  • the concentration of the cells of the mesenchymal cell lineage is at least 1, 2, 5, 10, 15 or 20 cells/cm 2 or about 10 cells/cm 2 .
  • the methods of the disclosure optionally include obtaining a population of cells enriched for cells of the mesenchymal cell lineage.
  • a cell population, or cell culture is enriched for cells of the mesenchymal cell lineage when it contains a greater percentage of cells of the mesenchymal cell lineage than a control cell population.
  • a control cell population is a cell population that has not been contacted with culture medium comprising a CSF1R inhibitor.
  • a cell population is enriched for cells of the mesenchymal cell lineage when it contains an increase in the number and/or size of mesenchymal cell colonies of compared to a control cell population.
  • a cell population is enriched for cells of the mesenchymal cell lineage when it contains at least 5%, 10%, 20%, 50% or 75% more mesenchymal cell colonies than a control cell population.
  • a cell population is enriched for cells of the mesenchymal cell lineage when the average size of the mesenchymal cell colonies is at least 5%, 10%, 20%, 50% or 75% larger than the average size of the mesenchymal cell colonies in a control cell population.
  • a cell population is enriched for cells of the mesenchymal cell lineage when it contains a decreased number of macrophages compared to a control cell population. In some options, a cell population is enriched for cells of the mesenchymal cell lineage when there is at least a 5%, 10%, 20%, 50% or 75% decrease in the number of macrophages in the cell population compared to a control cell population. In one option, the macrophages are C45+/CD11b+ macrophages.
  • a cell population is enriched for cells of the mesenchymal cell lineage when it contains fewer mature macrophages that have been differentiated from hematopoietic monocytes compared to a control cell population.
  • a cell population is enriched for cells of the mesenchymal cell lineage when macrophage differentiation is suppressed and hematopoetic monocytes are maintained.
  • a cell population may be enriched for cells of the mesenchymal cell lineage when there is at least a 5%, 10%, 20%, 50% or 75% increase in the number of hematopoetic monocytes in the cell population compared to a control cell population and/or at least a 5%, 10%, 20%, 50% or 75% decrease in the number of mature macrophages in the cell population compared to the control cell population.
  • Hematopoietic monocytes are optionally identified by a CD45+/CD11b+/Sca1+ phenotype.
  • Mature macrophages are optionally identified by a CD45+/CD11b+/Sca1- phenotype.
  • the present disclosure also provides culture media compositions useful for culturing cells of the mesenchymal cell lineage.
  • the culture media compositions are also useful for enriching a cell population for cells of the mesenchymal cell lineage.
  • the culture media comprises a CSF1R kinase inhibitor.
  • the CSF1R kinase inhibitor is GW2580, KI20227, HY-13075, cFMS Receptor Inhibitor II, cFMS Receptor Inhibitor III, cFMS Receptor Inhibitor IV or ARRY-382.
  • the media comprises 0.1 to 20 uM GW2580, 0.5 to 15 uM GW2580, 1 to 10 uM GW2580 or about 0.1 ⁇ M, 0.5 ⁇ M, 1 uM, 5 uM, 10uM, 15 uM or 20 uM GW2580.
  • the media comprises 0.1 to 20 uM KI20227, 0.5 to 15 uM KI20227, 1 to 10 uM KI20227 or about 0.1 ⁇ M, 0.5 ⁇ M, 1 uM, 5 uM, 10uM, 15 uM or 20 uM KI20227.
  • the culture media comprising a CSF1R kinase inhibitor allows the proliferation of cells of the mesenchymal cell lineage at a higher rate than culture media that does not comprise a CSF1R kinase inhibitor.
  • the culture media comprises at least one growth factor.
  • the growth factor is FGF, EGF, IGF or bFGF or any combination thereof.
  • the culture media can be any culture media useful in culturing cells of the mesenchymal cell lineage.
  • the culture media comprises a basal media.
  • Basal media are known in the art and include Dulbecco's modified Eagles's medium (DMEM), advanced DMEM, BiogroTM, SkGMTM, Ham's F10, Ham's F12, Iscove's modified Dulbecco's medium, neurobasal medium, RPMI 1640 and MCDB120 medium.
  • the culture media may be serum free or may contain serum.
  • the culture media comprises a supplement or a combination of supplement.
  • supplements include, but are not limited to insulin, transferring, selenite, B27, dexamethasone, insulin, fetuin and albumin and growth factors such as FGF, EGF, IGF or bFGF.
  • Specific ombinations of supplements that may be included in the media include, but are not limited to:
  • the culture medium further comprises a lipid.
  • lipids include, but are not limited to, arachidonic acid, cholesterol, DL- ⁇ -tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitoleic acid, palmitic acid and stearic acid.
  • Enriched populations of MSCs can be obtained according to the methods described herein.
  • Applications and uses of enriched populations of MSCs include, but are not limited to, expansion of MSCs, differentiation of MSCs into mesenchymal lineages such as osteogenic, chondrogenic, adipogenic, or myogenic lineages and differentiation of MSCs into non-mesenchymal lineages such as epithelial, cardiomyogenic, neural, or hepatogenic.
  • the differentiated lineages can be used for both in vitro or in vivo purposes.
  • Example 1 Obtaining MSCs from adult mice
  • mice For mouse cells, the procedure involves the use of hind limbs of freshly euthanized (e.g. from 0 to 24 hours after euthanization) adult mice as a source of BM and CB-derived MSC.
  • the hind legs of euthanized mice, including the hip bones are skinned and removed from the animals. Adjoining soft tissues and epiphyses are completely removed and scraped off the bones. Clean bones are then transferred to a mortar containing wash buffer (PBS, 2% FBS, 1 mM EDTA) and lightly crushed with a pestle to release the marrow.
  • the wash buffer containing the marrow is transferred to a tube and the process repeated until all the marrow has been removed and the buffer is clear of red blood cells.
  • the buffer containing the marrow is then spun down and P0 cells resuspended in expansion medium and plated at 6.7 x 10 5 cells/cm 2 (50 million cells per T75 flask) in the presence of 1 ⁇ M GW2580 for expansion of MSC, or at densities ranging from 2.5 - 10 x 10 5 cells/cm 2 in 6-well plates in the presence of 1 ⁇ M GW2580 for CFU-F assays.
  • the crushed bones in the mortar are then transferred to a petri dish containing 2 ml of collagenase and finely minced with a scalpel. Minced bones are then digested in collagenase at 37°C with 100 rpm circular motion for 1 hour.
  • Digested bones are passed through a 40 ⁇ m strainer to generate a single cell solution.
  • This strained solution is spun down at 300 x g for 5 minutes, and P0 cells are resuspended in expansion medium and plated at 2.7 x 10 4 cells/cm 2 (2 million cells per T75 flask) in the presence of 1 ⁇ M GW2580 for expansion of MSC, or at densities ranging from 5 - 25 x 10 3 cells/cm 2 in 6-well plates in the presence of 1 ⁇ M GW2580 for CFU-F assays.
  • the P0 expansion cultures of BM and CB are incubated at 37°C in a humidified chamber at 5% CO 2 5% O 2 for 10 to 14 days to allow for expansion and enrichment of MSC.
  • Cultures are then passaged and plated at 6.7 x 10 3 cells/cm 2 in the presence of 1 ⁇ M GW2580 until they reach 70 to 80% confluence. Passaging is repeated every 7 days up to 10 times or until enough enriched MSCs are obtained for downstream applications. CFU-F assay cultures are fixed and stained with Toluidine blue at day 14 for colony formation assessment. For comparison purposes, control cultures for expansion and CFU-F assays are set up in the presence of DMSO vehicle instead of GW2580.
  • Example 2 Obtaining MSCs from human cells
  • BM is often the preferred tissue for extraction and enrichment of MSC. These cells are obtained by Ficoll extraction to remove red blood cells. During this process, fresh BM is diluted 5:14 in isolation buffer (PBS + 2% FBS + 2 mM EDTA) and spun down at 300 x g for 30 minutes. The interface layer containing the mononuclear cells is then removed and resuspended in 40 ml cold isolation buffer, which is then centrifuged again at 300 x g for 10 minutes. The supernatant is removed and cells resuspended in 2 ml expansion medium and counted.
  • isolation buffer PBS + 2% FBS + 2 mM EDTA
  • P0 cells are then resuspended in expansion medium and plated at 6.7 x 10 5 cells/cm 2 (50 million cells per T75 flask) in the presence of 1 ⁇ M GW2580 for expansion of MSC, or at densities ranging from 2.5 - 10 x 10 5 cells/cm 2 in 6-well plates in the presence of 1 ⁇ M GW2580 for CFU-F assays.
  • Human BM cultures are incubated at 37°C in a humidified chamber containing 5% CO 2 for 10 to 14 days to allow for expansion and enrichment of MSC. Cultures are then passaged and plated at 6.7 x 10 3 cells/cm 2 in the presence of 1 ⁇ M GW2580 until they reach 70 to 80% confluence.
  • GW2580 [5-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine] belongs to a class of small molecules that specifically inhibits CSF1R kinase activity by competing with ATP binding to CSF1R kinase (11). It has been shown that GW2580 completely inhibits human CSF1R kinase at 60 nM while remaining inactive against 26 other kinases tested.
  • KI20227 ⁇ N- ⁇ 4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl ⁇ -N0-[1-(1,3-thiazole-2-yl)ethyl] urea ⁇ another CSF1R kinase inhibitor, was used to demonstrate that prevention of macrophage, differentiation, proliferation, and survival in BM and CB-derived cultures of MSC is not limited to GW2580, but instead, is an activity observed with this class of CSF1R inhibitor molecules.
  • GW2580 (C 20 H 22 N 4 O 3 ) was obtained from LC Labs or BioVision and its molecular weight is 366.41. Aliquots of stocks that were 1,000 times concentrated were prepared and kept at -20C for immediate fresh use or for stability testing as follows:
  • KI20227 (C 24 H 24 N 4 O 5 S) was obtained from Cedarlane and its molecular weight is 480.54. KI20227 was used at 1 ⁇ M and a 1,000 times stock was prepared as follows and stored at -20C:
  • Example 5 CFU-F assays of mouse BM-derived cells show an increase in number and size of MSC colonies and a reduction in macrophage contamination when cultures are exposed to 1 ⁇ M GW2580.
  • Mouse BM-derived cells were isolated as described previously and CFU-F assays setup as described above. Assays were maintained in medium containing 0.1% v/v DMSO as control or 1 ⁇ M GW2580 for 14 days. Cultures were then stained with Toluidine blue and colony formation and appearance assessed.
  • Example 6 CFU-F assay of mouse CB-derived cells shows an increase in number and size of MSC colonies and a reduction in macrophage contamination when cultures are exposed to 1 ⁇ M GW2580.
  • Mouse CB-derived cells were isolated as described previously and CFU-F assays setup as described above. Assays were maintained in medium containing 0.1% v/v DMSO as control or 1 ⁇ M GW2580 for 14 days. Cultures were then stained with Toluidine blue and colony formation and appearance assessed.
  • Example 7 Expansion of mouse cultures of both BM and CB-derived cells shows a significant reduction in the number of macrophage contamination when cultures are treated with 1 ⁇ M GW2580.
  • BM and CB-derived cultures were isolated as described above and expansion assays setup as described above. Assays were maintained in medium containing 0.1% v/v DMSO as control or 1 ⁇ M GW2580. Flow cytometry analysis was performed at P1 and images collected at P2.
  • Example 8 Continuous exposure of expansion cultures of mouse BM-derived cultures to 1 ⁇ M GW2580 for at least 3 passages leads to increased enrichment of the MSC population while greatly reducing differentiation of contaminating macrophages.
  • BM-derived cultures were isolated as described above and expansion assays setup as described above. Assays were maintained in medium containing 0.1% v/v DMSO as control or 1 ⁇ M GW2580 for passages P0 to P3. Images were acquired for at each passage and they show that control cultures are overgrown with contaminating macrophages at each passage time. Cultures treated with 1 ⁇ M GW2580, however, have reduced numbers of macrophages and most of the adherent cells in culture display a fibroblastic morphology typical of MSC ( Figure 5 ).
  • Example 9 Continuous exposure of expansion cultures of mouse CB-derived cultures to 1 ⁇ M GW2580 for at least 3 passages leads to increased enrichment of the MSC population while greatly reducing differentiation of contaminating macrophages.
  • CB-derived cultures were isolated as described above and expansion assays setup as described above. Assays were maintained in medium containing 0.1% v/v DMSO as control or 1 ⁇ M GW2580 for passages P0 to P3. Images were acquired at each passage and they show that control cultures are overgrown with contaminating macrophages at each passage time. Cultures treated with 1 ⁇ M GW2580, however, have reduced numbers of macrophages and most of the adherent cells in culture display a fibroblastic morphology typical of MSC ( Figure 8 ).
  • Example 10 Exposure of expansion cultures of mouse BM and CB-derived cells to increasing concentrations ranging from 1 to 10 ⁇ M GW2580 leads to increasing prevention of macrophage differentiation and increasing enrichment of MSC in culture in a concentration-dependent manner in CB-derived cultures, but not in BM-derived cultures.
  • BM and CB-derived cultures were isolated as described above and expansion assays setup as described above. Assays were maintained in medium containing 0.1% v/v DMSO as control or treated with 1, 5, or 10 ⁇ M GW2580. Images of cultures show that control cultures are overgrown with contaminating macrophages, while cultures treated with 1, 5, or 10 ⁇ M GW2580 have reduced numbers of contaminating macrophages. In addition, most of the adherent cells in both BM and CB-derived cultures display a healthy fibroblastic morphology typical of MSC when compared to control cultures ( Figure 11 ).
  • CD45 + /CD11b + /Sca1 + hematopoietic monocyte phenotype 93% of the CD45 + cells are CD11b + mature macrophages, and only 7% maintained a CD45 + /CD11b + /Sca1 + hematopoietic monocyte phenotype.
  • Cultures treated with 1, 5, or 10 ⁇ M GW2580 show a dose-response decrease in CD45 + /CD11b + mature macrophages from 75 to 60 and to 50%, respectively. This decrease in macrophage differentiation translated into a dose-response increase in CD45 + /CD11b + /Sca1 + hematopoietic monocytes from 18 to 32 and to 41%, respectively.
  • Example 11 Continuous treatment of expansion cultures of CB-derived cells with 1 ⁇ M GW2580 is necessary to prevent macrophage differentiation and proliferation.
  • CB-derived cultures were isolated as described above and expansion assays setup as described above. Assays were maintained in medium containing 1 ⁇ M GW2580 during passages P0 to P1 and then maintained in control medium containing 0.1% v/v DMSO during P2 to P3. At the end of P3, adherent and non-adherent cells were collected and flow cytometry analysis was performed as indicated above. Samples were gated on viable CD45 + cells and analysis of adherent and non-adherent cells demonstrated that 97% of the adherent cells and 99% of the non-adherent cells were CD45 + /CD11b + /Sca1 - mature macrophage. These results suggest that continuous exposure to 1 ⁇ M GW2580 is necessary to prevent macrophage differentiation in culture (compare Figure 14 to control treatment in Figure 10 ).
  • CB-derived cultures were isolated as described above and expansion assays setup as described above. Cultures were maintained in control medium containing 0.1% v/v DMSO during passages P0 to P2 and then maintained in medium containing 1 ⁇ M GW2580 during P3 to P4. For comparative purposes, P4 control cultures were enriched for mesenchymal progenitors using the EasySep Isolation of Mesenchymal Progenitors From Mouse Compact Bone (STEMCELL catalogue # 19771) as per manufacturer's instruction. Cells were collected and flow cytometry analysis was performed as indicated in above. Samples were gated on viable CD45 + cells, which in GW2580-treated and EasySep-enriched samples comprised ⁇ 0.2% of total cells.
  • CD45 + cells only around 25% of the cells were CD11b + /Sca1 - mature macrophages in both samples. In addition, in both samples about 75% of the CD45 + cells were CD11 + /Sca1 + hematopoietic monocytes ( Figure 15 ).
  • BM and CB-derived cultures were isolated as described above and expansion assays setup as described above. Cultures were maintained in medium containing 1 ⁇ M KI20227 or in medium containing 1 ⁇ M GW2580 up to P2. Cells were collected and flow cytometry analysis was performed as indicated above. Samples were gated on viable cells, and analysis show that in GW2580-treated cultures, ⁇ 1% of the cells were CD45 + /CD11b + mature macrophages. In KI20227-treated cultures, ⁇ 2% were CD45 + /CD11b + mature macrophages ( Figure 16 ).
  • Example 14 Treatment of human BM-derived MSC cultures with 1 ⁇ M GW2580 does not hinder the differentiation potential of these cells.
  • BM-derived cultures were isolated as described above and expansion assays setup as described above. These P2 expansion cultures were maintained in control medium containing 0.1% v/v DMSO or in medium containing 1 ⁇ M GW2580. At the end of P2, cell were dissociated by trypsin digestion and replated at 3 x 10 4 cells/cm 2 in 12-well plates for differentiation assays. Differentiation cultures were maintained for 3 weeks in adipogenic or osteogenic differentiation media. At the end of the 3-week period, cultures were fixed and stained with Oil red O to assess adipogenic differentiation, or with silver nitrate to assess osteogenic differentiation. Cultures that were expanded in the presence of 1 ⁇ M GW2580 show similar levels of adipogenic and osteogenic differentiation as cultures that were expanded in control medium ( Figure 17 ).
  • Example 15 CFU-F assays of BM- and CB-MSC exposed to GW2580 for 14 days show an increase in the number of MSC colonies in culture.
  • CFU-F assays for both BM- and CB-MSC were setup as in Example 4a and 4b.
  • assay 1 cultures were exposed to 1 uM GW2580 or vehicle control.
  • assays 2 and 3 cultures were exposed to 2.5 uM GW2580 or vehicle control. These assays were maintained for 14 days without media change. At the end of day 14, cultures were fixed and stained with toluidine blue and the number of MSC colonies counted.
  • Example 16 BM- and CB-MSC cultures show a significant enrichment in CD45 - cells with an increase in CD29 + /Sca1 + cells when exposed to 2.5 uM GW2580 for 14 days during P0.
  • Example 17 Exposure of BM- and CB-MSC cultures to GW2580 during expansion facilitates downstream differentiation of these cells and requires fewer cells than cultures exposed to a vehicle control
  • BM-MSC cultures were maintained in control medium or in medium containing GW2580 for 2 passages and then plated at different densities for differentiation into the osteogenic and adipogenic lineages ( Fig. 18 ).
  • BM-MSCs that were initially exposed to vehicle- or GW2580-containing media were plated at 3 different densities ( Fig. 18A ) in MesenCult Proliferation Medium and allowed to adhere to the plates overnight.
  • both BM- and CB-MSC were expanded for 2 passages in the presence of 2.5 uM GW2580 and then plated at 4.0 x 10 4 cells/cm 2 . Cultures were then differentiated for 14 days into the adipogenic and osteogenic lineages ( Fig. 18B ). A substantial adipogenic and osteogenic differentiation was observed in these cultures as demonstrated by Oil Red O and ALP/mineralization staining, respectively. Taken together, these data suggest that exposure of BM- and CB-MSC cultures to GW2580 facilitates their ability to strongly differentiate into different lineages, likely due to the enrichment and increased proliferation of MSC observed in cultures exposed to GW2580 when compared to control cultures.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Rheumatology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Claims (11)

  1. Procédé in vitro de culture de cellules, ledit procédé comprenant :
    (a) la mise en contact d'une population cellulaire comprenant des cellules de la lignée cellulaire mésenchymateuse et des cellules non mésenchymateuses avec un milieu de culture comprenant un inhibiteur de kinase CSF1R ; et,
    (b) l'enrichissement de la population cellulaire par des cellules de la lignée cellulaire mésenchymateuse, dans lequel la population cellulaire contient au moins 5 % plus de colonies de cellules mésenchymateuses qu'une population cellulaire témoin,
    dans lequel l'inhibiteur de kinase CSF1R est le GW2580, le KI20227, le HY-13075, l'inhibiteur de récepteur cFMS II, l'inhibiteur de récepteur cFMS III, l'inhibiteur de récepteur cFMS IV ou l'ARRY-382, et dans lequel les cellules de la lignée cellulaire mésenchymateuse comprennent au moins une cellule souche mésenchymateuse et un progéniteur de cellules mésenchymateuses.
  2. Procédé selon la revendication 1, dans lequel le procédé comprend :
    a) la récolte de cellules à partir d'un échantillon de tissu obtenu d'un sujet, dans lequel les cellules récoltées comprennent des cellules de la lignée cellulaire mésenchymateuse, et
    b) la mise en contact des cellules récoltées avec le milieu de culture comprenant l'inhibiteur de kinase CSF1R ; et éventuellement
    c) l'obtention d'une population cellulaire enrichie pour les cellules de la lignée cellulaire mésenchymateuse.
  3. Procédé selon la revendication 2, dans lequel l'échantillon de tissu comprend une moelle osseuse, un os compact ou un tissu adipeux.
  4. Procédé selon l'une quelconque des revendications 1 à 3, dans lequel les cellules sont mises en contact avec le milieu de culture pendant au moins une heure, au moins un jour ou au moins une semaine.
  5. Procédé selon l'une quelconque des revendications 1 à 4, dans lequel les cellules de la lignée cellulaire mésenchymateuse conservent leur capacité à former des lignées cellulaires adipogènes, chrondrogéniques et ostéogènes.
  6. Milieu de culture cellulaire comprenant un inhibiteur de kinase CSF1R, dans lequel l'inhibiteur de kinase CSF1R est le GW2580, le KI20227, le HY-13075, l'inhibiteur du récepteur cFMS II, l'inhibiteur du récepteur cFMS III, l'inhibiteur du récepteur cFMS IV ou le ARRY-382, le milieu comprenant en outre une combinaison de facteurs de croissance choisis dans la liste constituée par FGF, EGF et IGF, et dans lequel le milieu comprend en outre un lipide.
  7. Milieu de culture cellulaire selon la revendication 6, dans lequel le milieu comprend un milieu basal.
  8. Milieu de culture cellulaire selon la revendication 7, dans lequel le milieu basal est choisi parmi le milieu d'eagle modifié par dulbecco (DMEM), le DMEM avancé, le Biogro™, le SkGM™, le F10 de Ham, le F12 de Ham, le milieu de dulbecco modifié par le milieu d'iscove, le milieu neurobasal, le RPMI 1640 ou le MCDB120.
  9. Milieu de culture cellulaire selon l'une quelconque des revendications 6 à 8, le milieu comprenant en outre un supplément, de préférence choisi parmi :
    • l'insuline, le transfert et le sélénite (ITS) ;
    • le B27 ;
    • la dexaméthasone, l'insuline, l'EGF, la fétuine et l'albumine ; et
    • la dexaméthasone, le bFGF, l'albumine et l'insuline.
  10. Milieu de culture cellulaire selon l'une quelconque des revendications 6 à 9, dans lequel le lipide est l'acide arachidonique et/ou le cholestérol et/ou l'acétate de DL-a-tocophérol et/ou l'acide linoléique et/ou l'acide linolénique et/ou l'acide myristique et/ou l'acide oléique et/ou l'acide palmitoléique et/ou l'acide palmitique et/ou l'acide stéarique.
  11. Milieu de culture cellulaire selon l'une quelconque des revendications 6 à 10, le milieu étant exempt de sérum.
EP14765588.0A 2013-03-15 2014-03-12 Compositions et procédés pour obtenir des cultures de cellules souches mésenchymateuses enrichies Active EP2970905B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361789530P 2013-03-15 2013-03-15
PCT/CA2014/000216 WO2014138888A1 (fr) 2013-03-15 2014-03-12 Compositions et procédés pour obtenir des cultures de cellules souches mésenchymateuses enrichies

Publications (3)

Publication Number Publication Date
EP2970905A1 EP2970905A1 (fr) 2016-01-20
EP2970905A4 EP2970905A4 (fr) 2016-09-07
EP2970905B1 true EP2970905B1 (fr) 2019-10-16

Family

ID=51535661

Family Applications (1)

Application Number Title Priority Date Filing Date
EP14765588.0A Active EP2970905B1 (fr) 2013-03-15 2014-03-12 Compositions et procédés pour obtenir des cultures de cellules souches mésenchymateuses enrichies

Country Status (7)

Country Link
US (1) US10017741B2 (fr)
EP (1) EP2970905B1 (fr)
CN (2) CN105026553A (fr)
AU (1) AU2014231709A1 (fr)
CA (1) CA2906132C (fr)
IL (1) IL240871B (fr)
WO (1) WO2014138888A1 (fr)

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3000875A1 (fr) * 2014-09-25 2016-03-30 Philipps-Universität Marburg Procédé de fabrication d'une composition comprenant des cellules stromales mésenchymateuses humaines expansées ex vivo
US20180245048A1 (en) * 2015-02-25 2018-08-30 Agency For Science, Technology And Research Methods and compositions for expansion and differentiation of skeletal muscle stem cells or progenitor cells
JP7154586B2 (ja) * 2016-02-23 2022-10-18 ユニバーシティ－インダストリー コーオペレイション グループ オブ キョンヒ ユニバーシティ 幹細胞の効能改善のための組成物及び方法
MA44611A (fr) * 2016-04-04 2019-02-13 Massachusetts Inst Technology Compositions de composés hydrophobes cristallisés et leurs procédés de préparation et d'utilisation
US20190292246A1 (en) 2016-11-03 2019-09-26 Juno Therapeutics, Inc. Combination therapy of a cell based therapy and a microglia imhibitor
AU2018275894B2 (en) 2017-06-02 2025-04-24 Juno Therapeutics, Inc. Articles of manufacture and methods for treatment using adoptive cell therapy
CN111201438A (zh) 2017-06-02 2020-05-26 朱诺治疗学股份有限公司 与和细胞疗法相关的毒性有关的制品和方法
EP3644721A1 (fr) 2017-06-29 2020-05-06 Juno Therapeutics, Inc. Modèle murin pour évaluer des toxicités associées à des immunothérapies
WO2019046832A1 (fr) 2017-09-01 2019-03-07 Juno Therapeutics, Inc. Expression génique et évaluation d'un risque de développement d'une toxicité suite à une thérapie cellulaire
US11564946B2 (en) 2017-11-01 2023-01-31 Juno Therapeutics, Inc. Methods associated with tumor burden for assessing response to a cell therapy
CA3084013A1 (fr) 2017-12-01 2019-06-06 StemRIM Inc. Cellules souches mesenchymateuses ectodermiques et leur procede de production
WO2019156137A1 (fr) 2018-02-08 2019-08-15 株式会社ステムリム Agent thérapeutique contre le psoriasis
EP3833762A4 (fr) 2018-08-09 2022-09-28 Verseau Therapeutics, Inc. Compositions oligonucléotidiques pour cibler ccr2 et csf1r et leurs utilisations
US12304933B2 (en) 2018-10-05 2025-05-20 StemRIM Inc. Disease treatment drug based on mesenchymal-stem-cell mobilization
PL3886894T3 (pl) 2018-11-30 2024-07-01 Juno Therapeutics, Inc. Metody dawkowania i leczenia nowotworów b-komórkowych w adoptywnej terapii komórkowej
PL3886875T3 (pl) 2018-11-30 2024-09-09 Juno Therapeutics, Inc. Metody leczenia z wykorzystaniem adoptywnej terapii komórkowej
IL293393A (en) 2019-12-06 2022-07-01 Juno Therapeutics Inc Cell therapy-related toxicity and response-related methods for the treatment of b-cell malignancies
CN113215085B (zh) * 2021-05-07 2024-05-10 澳门大学 一种脂类物质添加剂及其应用
CN114058585A (zh) * 2021-12-03 2022-02-18 无锡市第二人民医院 一种小鼠骨髓来源树突状细胞的培养方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050265980A1 (en) * 2004-05-14 2005-12-01 Becton, Dickinson And Company Cell culture environments for the serum-free expansion of mesenchymal stem cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
CN120005816A (zh) 2025-05-16
CN105026553A (zh) 2015-11-04
CA2906132A1 (fr) 2014-09-18
US20160032248A1 (en) 2016-02-04
US10017741B2 (en) 2018-07-10
EP2970905A1 (fr) 2016-01-20
IL240871B (en) 2020-05-31
WO2014138888A1 (fr) 2014-09-18
EP2970905A4 (fr) 2016-09-07
CA2906132C (fr) 2021-01-05
IL240871A0 (en) 2015-10-29
AU2014231709A1 (en) 2015-10-15

Similar Documents

Publication Publication Date Title
EP2970905B1 (fr) Compositions et procédés pour obtenir des cultures de cellules souches mésenchymateuses enrichies
Lindroos et al. Serum-free, xeno-free culture media maintain the proliferation rate and multipotentiality of adipose stem cells in vitro
Parker et al. Low serum and serum-free culture of multipotential human adipose stem cells
Stolzing et al. Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies
JP2022046511A (ja) 胎盤幹細胞を使用する免疫調節
US10105399B2 (en) Methods and compositions for treatment of bone defects with placental cell populations
JP6348848B2 (ja) 間葉系幹細胞の増殖
CN103060263B (zh) 胎盘干细胞群
Choudhery et al. Cryopreservation of whole adipose tissue for future use in regenerative medicine
CA2856764C (fr) Composition de milieu de culture pour rajeunir des cellules souches
Li et al. An efficient and economical way to obtain porcine muscle stem cells for cultured meat production
US20210032596A1 (en) Method of producing erythrocytes
Esmaeili et al. The impact of parathyroid hormone treated mesenchymal stem cells on ex-vivo expansion of cord blood hematopoietic stem cells
US9687512B2 (en) Isolated cardiac stem cells and methods of their use
US20150164947A1 (en) Novel adult progenitor cell
RU2722669C1 (ru) Способ получения ассоциатов гемопоэтических и стромальных клеток-предшественников, способных подавлять активацию и пролиферацию аллогенных лимфоцитов
Fereydani et al. Ex vivo expansion of hematopoietic stem cells in two/three-dimensional co-cultures with various source of stromal cells
KR100702862B1 (ko) 제대혈 유래 간엽줄기세포를 세포영양막으로 이용한조혈모세포/전구세포의 체외 성장 및 증식방법
Guan et al. In vitro differentiation of human skin-derived mesenchymal stem cells into lymphocytes: Possibility evaluation
Willemze et al. Preferential chondrogenic differentiation potential of human bone marrow-derived mesenchymal stem cells
HK1155775B (en) Novel adult progenitor cell
HK1166515A (en) Methods and compositions for treatment of bone defects with placental cell populations
HK1166997A (en) Methods and compositions for treatment of bone defects with placental cell populations

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20151008

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

RIN1 Information on inventor provided before grant (corrected)

Inventor name: DURONIO, CHRISTOPHER

Inventor name: SHORT, BRENTON JOHN

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20160808

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 5/077 20100101AFI20160802BHEP

Ipc: C12N 5/02 20060101ALI20160802BHEP

Ipc: C12N 5/0775 20100101ALI20160802BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20170517

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20190506

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602014055284

Country of ref document: DE

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1191269

Country of ref document: AT

Kind code of ref document: T

Effective date: 20191115

REG Reference to a national code

Ref country code: NL

Ref legal event code: FP

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1191269

Country of ref document: AT

Kind code of ref document: T

Effective date: 20191016

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200217

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200116

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200117

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200116

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200224

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602014055284

Country of ref document: DE

PG2D Information on lapse in contracting state deleted

Ref country code: IS

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200216

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

26N No opposition filed

Effective date: 20200717

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20200331

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200312

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200331

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200331

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200312

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200331

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191016

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230511

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 20250314

Year of fee payment: 12

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20250314

Year of fee payment: 12

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20260323

Year of fee payment: 13

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20260326

Year of fee payment: 13

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20260323

Year of fee payment: 13